Lupus afflicts women 9 times more often than men. Estrogen contributes to lupus severity, but does not completely explain the increased risk. Impaired DNA methylation, a repressive epigenetic modification, causes overexpression of T cell genes that contribute to lupus. DNA methylation also silences one X chromosome in women. CD40LG is an X-linked gene known to be overexpressed in lupus and contribute to autoantibody production. We found that CD40LG on the inactive X demethylates and is overexpressed in women but not men with lupus, predisposing women to lupus. Other X-linked genes may also demethylate, predisposing to autoimmunity. We surveyed methylation sensitive T cell genes, and identified O-linked N-acetylglucosamine transferase (OGT) as another X-linked gene overexpressed in demethylated female T cells. OGT couples N-acetylglucosamine (GlcNAc) to serines and threonines in a variety of proteins including signaling molecules, modifying function in a manner analogous to phosphorylation and referred to as the hexosamine signaling pathway (HSP). HSP abnormalities are implicated in diabetes and neurodegeneration, but little is known regarding its role in T cells. We hypothesize that demethylation of OGT on the inactive X results in overexpression in women, altering HSP signaling and contributing to pathogenic T cell function by modifying signaling. We will test this hypothesis by: 1) Comparing OGT mRNA and protein levels in control and demethylated CD4+ and CD8+ T cells from healthy men and women with levels in CD4+ and CD8+ T cells from men and women with inactive lupus, active lupus, and disease/age controls, and confirming OGT demethylation by bisulfite sequencing, 2) Determining if OGT overexpression impairs T cell ERK, JNK and/or p38 pathway signaling and modifies T cell gene expression patterns, and 3) Determining the functional significance of T cell OGT overexpression on the development of autoimmunity using transgenic mice with a T cell specific inducible OGT transgene. These studies will characterize a new pathway regulating T cell gene expression, and characterize how abnormalities in the pathway may predispose women to autoimmunity. PUBLIC HEALTH RELEVANCE: Lupus is an autoimmune disease primarily affecting women, causing disability and sometimes death. The reason women are predisposed to lupus is unknown. Our group has found that a mechanism regulating gene expression, called DNA methylation, is defective in T lymphocytes from lupus patients, causing overexpression of genes that make the cells attack the body. This contributes to lupus in those with the appropriate genetic makeup. Women have 2 X chromosomes, while men have only one. One X chromosome is inactivated by DNA methylation in women to prevent them from expressing the genes twice as much as men. However, genes on the second X chromosome demethylate in women with lupus, causing overexpression of genes that may contribute to their disease. We have found that OGT, an X chromosome gene important in regulating cellular function, doubles expression in T lymphocytes from women when DNA methylation is inhibited. This suggests that OGT may also be overexpressed in women with lupus, contributing to their disease. The studies described in this application will determine if women with lupus overexpress OGT, and determine the functional consequences. The results may provide new insights into mechanisms causing autoimmunity in women, and suggest new ways to treat the disease.